The teratogenic activity of valproate (VPA), an antiepileptic and an inhibitor of histone deacetylase (HDACi), is dose-dependent in humans. Previous results showed that VPA impairs in vitro ...development and neural differentiation of the gastrulating embryo proper. We aimed to investigate the impact of a lower VPA dose in vitro and whether this effect is retained in transplants in vivo. Rat embryos proper (E9.5) and ectoplacental cones were separately cultivated at the air-liquid interface with or without 1 mM VPA. Embryos were additionally cultivated with HDACi Trichostatin A (TSA), while some cultures were syngeneically transplanted under the kidney capsule for 14 days. Embryos were subjected to routine histology, immunohistochemistry, Western blotting and pyrosequencing. The overall growth of VPA-treated embryos in vitro was significantly impaired. However, no differences in the apoptosis or proliferation index were found. Incidence of the neural tissue was lower in VPA-treated embryos than in controls. TSA also impaired growth and neural differentiation in vitro. VPA-treated embryos and their subsequent transplants expressed a marker of undifferentiated neural cells compared to controls where neural differentiation markers were expressed. VPA increased the acetylation of histones. Our results point to gastrulation as a sensitive period for neurodevelopmental impairment caused by VPA.
Antioxidant N-tert-Butyl-α-phenylnitron (PBN) partly protected embryos from the negative effects of a DNA demethylating drug 5-azacytidine during pregnancy. Our aim was to investigate PBN's impact on ...the placenta. Fischer rat dams were treated on gestation days (GD) 12 and 13 by PBN (40 mg/kg), followed by 5azaC (5 mg/kg) after one hour. Global methylation was assessed by pyrosequencing. Numerical density was calculated from immunohistochemical expression in single cells for proliferating (PCNA), oxidative (oxoguanosine) and nitrosative (nitrotyrosine) activity. Results were compared with the PBN-treated and control rats. PBN-pretreatment significantly increased placental weight at GD15 and GD20, diminished by 5azaC, and diminished apoptosis in GD 20 placentas caused by 5azaC. Oxoguanosine expression in placentas of 5azaC-treated dams was especially high in the placental labyrinth on GD 15, while PBN-pretreatment lowered its expression on GD 15 and GD 20 in both the labyrinth and basal layer. 5azaC enhanced nitrotyrosine level in the labyrinth of both gestational stages, while PBN-pretreatment lowered it. We conclude that PBN exerted its prophylactic activity against DNA hypomethylating agent 5azaC in the placenta through free radical scavenging, especially in the labyrinthine part of the placenta until the last day of pregnancy.
Background
The incretin hormone glucagon‐like peptide‐l (GLP‐1) is an important regulator of post‐prandial insulin secretion, acting through a G protein‐coupled cell surface receptor (GLP‐1R). In ...addition to its expression in pancreatic β‐cells, several studies suggested that GLP‐1R is located in extra‐pancreatic tissues.
Objectives
In this study, we examined for the first time the testicular distribution of the GLP‐1R, both in normal human and neoplastic testicular tissues as well as in rodent testis and rodent testicular cell lines.
Methods and Methods
The GLP‐1R distribution in testicular section has been evaluated by immunohistochemistry, the specificity of IHC was validated by demonstrating a positive staining for GLP‐1RmRNA by RISH technology. While GLP‐1R expression in terms of protein was detected by western blot analysis, Moreover, mRNA levels were determined in human testis, in rodent Leydig, and Sertoli cell lines.
Results
Using immunohistochemistrya specific staining for GLP‐1R was detected in Leydig cells. The specificity of IHC was validated by demonstrating a positive staining for GLP‐1RmRNA only in these cell types. Species differences in the GLP‐1R expression between humans and rodents were observed. Interestingly, a decreased expression of the receptor in rodent tumor Leydig cell line and an absence in human Leydig tumor samples was detected.
Discussion
It may be hypothesized that GLP‐1R acts like an oncosuppressor in Leydig tumors. A role in regulation of hormone secretion by GLP‐1 has been shown in other endocrine cells, therefore we hypothesized that GLP‐1R is able to modulate somehow the Leydig cell function.
Conclusion
In our findings, a careful evaluation of human testicular tissues and rodent testis revealed Leydig cells as a potential target for GLP‐1. Collectively, an effect of GLP‐1R in Leydig cell function may be presumed although future studies are needed to ascertain the GLP‐1R’s role both in normal and tumor Leydig cells.
The increase in cancer survival rates has put a focus on ensuring fertility preservation procedures for cancer patients. Ovarian tissue cryopreservation presents the only option for prepubertal girls ...and patients who require immediate start of treatment and, therefore, cannot undergo controlled ovarian stimulation. We aimed to provide an assessment of stem cells' impact on cryopreserved ovarian tissue grafts in regard to the expression of growth factors, angiogenesis promotion, tissue oxygenation, ovarian follicle survival and restoration of endocrine function. For this systematic review, we searched the Scopus and PubMed databases and included reports of trials using murine and/or human cryopreserved ovarian tissue for transplantation or in vitro culture in combination with mesenchymal stem cell administration to the grafting site. Of the 1201 articles identified, 10 met the criteria. The application of stem cells to the grafting site has been proven to support vascular promotion and thereby shorten the period of tissue hypoxia, which is reflected in the increased number of remaining viable follicles and faster recovery of ovarian endocrine function. Further research is needed before implementing the use of stem cells in OT cryopreservation and transplantation procedures in clinical practice. Complex ethical dilemmas make this process more difficult.
Azoospermia is a form of male infertility characterized by a complete lack of spermatozoa in the ejaculate. Sertoli cell-only syndrome (SCOS) is the most severe form of azoospermia, where no germ ...cells are found in the tubules. Recently, FANCM gene variants were reported as novel genetic causes of spermatogenic failure. At the same time, FANCM variants are known to be associated with cancer predisposition. We performed whole-exome sequencing on a male patient diagnosed with SCOS and a healthy father. Two compound heterozygous missense mutations in the FANCM gene were found in the patient, both being inherited from his parents. After the infertility assessment, the patient was diagnosed with diffuse astrocytoma. Immunohistochemical analyses in the testicular and tumor tissues of the patient and adequate controls showed, for the first time, not only the existence of a cytoplasmic and not nuclear pattern of FANCM in astrocytoma but also in non-mitotic neurons. In the testicular tissue of the SCOS patient, cytoplasmic anti-FANCM staining intensity appeared lower than in the control. Our case report raises a novel possibility that the infertile carriers of FANCM gene missense variants could also be prone to cancer development.
Seminoma (SE) is the most frequent type of testicular tumour, affecting predominantly young men. Early detection and diagnosis of SE could significantly improve life quality and reproductive health ...after diagnosis and treatment. Copy number variation (CNV) has already been associated with various cancers as well as SE. In this study, we selected four genes (
,
,
, and
) for CNV analysis in genomic DNA (gDNA), which are located on chromosomes susceptible to gains, and whose aberrant expression was already detected in SE. Furthermore, CNV was analysed in cell-free DNA (cfDNA) from seminal plasma. Analysis was performed by droplet digital polymerase chain reaction (ddPCR) on gDNA from SE and nonmalignant testicular tissue. Seminal plasma cfDNA from SE patients before and after surgery was analysed, as well as from healthy volunteers. The CNV hotspot in gDNA from SE tissue was detected for the first time in all analysed genes, and for two genes,
and
it was reflected in cfDNA from seminal plasma. Although clinical value is yet to be determined, presented data emphasize a potential use of CNV as a potential SE biomarker from a liquid biopsy.
Although DNA methylation epigenetically regulates development, data on global DNA methylation during development of limb buds (LBs) are scarce. We aimed to investigate the global DNA methylation ...developmental dynamics in rat LBs cultivated in a serum-supplemented (SS) and in chemically defined serum- and protein-free (SF) three-dimensional organ culture. Fischer rat front- and hind-LBs at 13th and 14th gestation days (GD) were cultivated at the air-liquid interface in Eagle's Minimal Essential Medium (MEM) or MEM with 50% rat serum for 14 days, as SF and SS conditions, respectively. The methylation of repetitive DNA sequences (SINE rat ID elements) was assessed by pyrosequencing. Development was evaluated by light microscopy and extracellular matrix glycosaminoglycans staining by Safranin O. Upon isolation, weak Safranin O staining was present only in more developed GD14 front-LBs. Chondrogenesis proceeded well in all cultures towards day 14, except in the SF-cultivated GD13 hind-LBs, where Safranin O staining was almost absent on day 3. That was associated with a higher percentage of DNA methylation than in SF-cultivated GD13 front-LBs on day three. In SF-cultivated front-LBs, a significant methylation increase between the 3rd and 14th day was detected. In SS-cultivated GD13 front-LBs, methylation increased significantly on day three and then decreased. In older GD14 SS-cultivated LBs, there was no increase of DNA methylation, but they were significantly hypomethylated relative to the SS-cultivated GD13 at days 3 and 14. We confirmed that the global DNA methylation increase is associated with less developed limb organ primordia that strive towards differentiation in vitro, which is of importance for regenerative medicine strategies.
Lacrimal gland dysfunction causes dry eye disease (DED) due to decreased tear production. Aqueous-deficient DED is more prevalent in women, suggesting that sexual dimorphism of the human lacrimal ...gland could be a potential cause. Sex steroid hormones are a key factor in the development of sexual dimorphism. This study aimed to quantify estrogen receptor (ER) and androgen receptor (AR) expression in the human lacrimal gland and compare it between sexes. RNA was isolated from 35 human lacrimal gland tissue samples collected from 19 cornea donors. AR, ERα, and ERβ mRNA was identified in all samples, and their expression was quantified using qPCR. Immunohistochemical staining was performed on selected samples to evaluate protein expression of the receptors. ERα mRNA expression was significantly higher than the expression of AR and ERβ. No difference in sex steroid hormone (SSH) receptor mRNA expression was observed between sexes, and no correlation was observed with age. If ERα protein expression is found to be concordant with mRNA expression, it should be investigated further as a potential target for hormone therapy of DED. Further research is needed to elucidate the role of sex steroid hormone receptors in sex-related differences of lacrimal gland structure and disease.
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